Mini trans blot electrophoretic transfer cell

MSRA proteins were isolated using an extraction kit (iNtRON Biotechnology, Kirkland, WA, USA), which included Tris-HCI (pH 7.5). All of the protein concentrations were extracted using a Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). The supernatant was removed, and proteins were transferred to fresh tubes. Proteins were separated utilizing SDS-PAGE and subsequently transferred onto nitrocellulose membranes (Millipore, MA, USA) for 3 h at 250 mA at 4 °C using the Bio-Rad electroblotting system (Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell). To block all unreacted holes in the membrane, a solution consisting of 5% skim milk in Tris-buffered saline and Tween-20 buffer was applied. The membranes were then probed with monoclonal mouse anti-PBP2a primary antibody and β-actin (diluted 1:1000; Bio-Rad, USA), then re-probed with anti-mouse IgG secondary antibody (diluted 1:2000, Enzo Life Sciences, Ann Arbor, MI, USA). Following the treatment of the membranes with ECL Prime Western Blotting Detection Reagent (Invitrogen, Waltham, MA, USA), an Image Quant LAS-4000 mini chemical luminescent imager (GE Healthcare Life Sciences, Issaquah, WA, USA) was used to observe the bands [22 (link)].

Total protein was extracted from treated cells using the RIAP lysis buffer (ThermoFisher, USA), and proteins electrophoresis were carried out using vertical Bio-Rad Mini-Protean II electrophoresis unit. Equal amounts of proteins were subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). Proteins were separated under reducing conditions for 3 h at 100 V and then separated proteins were transferred onto nitrocellulose membranes (Millipore, MA, USA) using Bio-Rad electroblotting system (Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell) for 3 h at 250 mA at 4°C. The membranes were blocked with 30 ml of 5% fat-skim milk in TBS containing 0.05% Tween-20 for 1 h at RT. Next, membranes were individually incubated for 1 h at RT with primary antibodies, mouse monoclonal anti-K-Ras, rabbit monoclonal anti-B-Raf, and mouse monoclonal anti-pho-P53. Finally, the membranes were washed 3 times with TBS-0.05% Tween-20 and then incubated with goat anti-rabbit secondary antibody conjugated with horseradish peroxidase. Signals were detected by the enhanced chemiluminescence system (ECL, Amersham), after soaking the membrane in the chemiluminescence solutions 1 and 2 (1:1) for minutes (26 (link)).

Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide gels and were subsequently subjected to western blotting. After SDS-PAGE, the proteins were blotted onto a polyvinylidene fluoride (PVDF) membrane with a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA). For western blotting, after blocking with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween 20 (TBST, pH 7.6), the membrane was incubated in either a 1,000-fold dilution of a rabbit polyclonal antibody against MGAT2 (hGnT II) (GeneTex, Los Angeles, CA, USA) or a 1,000-fold dilution of rabbit IgG B4GALT1 (hGalT I) (EnoGene Biotech, New York, NY, USA). The membrane was subsequently washed with TBST and then was incubated for 1 h in a 1:10,000 dilution of either an anti-mouse or anti-rabbit IgG antibody labeled with horseradish peroxidase (GE Healthcare Japan, Tokyo, Japan). Detection was performed by using ECL Plus western blotting reagent (GE Healthcare Japan). Specific bands were detected on a Fluor-S MAX MultiImager (Bio-Rad). For the lectin blots, the PVDF membrane was incubated with 2 μg/ml FITC-conjugated RCA120 (J-OIL MILLS, Tokyo, Japan), then washed with TBST. Specific fluorescent bands were detected with a Molecular Imager FX system (Bio-Rad).